Project Summary Each year, 20,000 people are diagnosed with myelodysplastic syndromes. Due to age and comorbidities, the majority of patients are ineligible for stem cell transplant, which could relieve them of disease. Instead these patients rely on blood transfusions and chemotherapy treatments to control their symptoms. 80% of these patients harbor mutations or deletions in genes that encode splicing factors, including U2AF1, SF3B and SRSF2. It has been shown that cells harboring mutations in these splicing factors display altered splicing patterns of multiple transcripts, and it is hypothesized that the dysregulation of these transcripts is involved in the pathogenesis of MDS. LUC7L2, an ortholog of yeast splicing factor LUC7, was recently identified as having decreased expression in 14% of MDS patients. Low expression of LUC7L2 is correlated with decreased patient survival, and genetic lesions in LUC7L2 results in differential alternative splicing of at least 44 genes, as shown by RNA-seq of patient samples. We hypothesize that LUC7L2 is involved regulates the splicing of a subset of transcripts and tumors cells with low levels of LUC7L2 have misregulated transcripts, which contributes to the pathogenesis of MDS. We will test this hypothesis using in vitro splicing assays to understand the effects of LUC7L2-deficiency on the process of splicing. We will also establish the RNA binding site of LUC7L2 using CLIP-Seq and RNA Bind-N- Seq. We will then determine which transcripts are actively regulated by LUC7L2 by performing RNA-Seq on multiple disease-relevant, LUC7L2-knockdown cell lines compared to wild type as well as patient samples and LUC7L2 knockout iPS cells. These experiments will be used to generate a list of transcripts that are bound and regulated by LUC7L2 that may contribute to oncogenesis. These transcripts will be tested for contribution to MDS phenotypes through a genetic rescue screen. Transcripts found to contribute to MDS will be individually validated in multiple disease-relevant cell models. The overall goal of this project is to identify previously unsuspected genes that are driving the pathogenesis of MDS by being dysregulated during splicing, and to provide targets for more specific therapies in the MDS patient population.